Rapid Peak Alignment for MALDI-TOF Lipid Analysis.

The ability to measure structural and functional alterations in cellular and tissue lipids with small footprint, accessible instrumentation has sparked interest in their role in disease pathology. However, various lipidomic analytical tools tend to be cumbersome and time-consuming. A rapid, accurate, and straight forward peak alignment software routine would greatly facilitate the analysis of large datasets, such as those produced by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Herein, we describe a novel Rapid Peak Alignment Method (RPAM) which allows untargeted analysis of lipids expressed in brain white matter following chronic ethanol exposure in an established experimental model. The RPAM outputs data comparable to manual peak alignments but the processing time requires only 90 minutes instead of 8-10 hours. This method is readily adapted to a broad range of models, tissue types, and human diseases.

The role of inflammation in the association between gestational diabetes and obstructive sleep apnea: A pilot study.

BACKGROUND: Obstructive sleep apnea is associated with pregnancy complications including gestational diabetes. Mechanisms underlying the association between obstructive sleep apnea and gestational diabetes remain to be elucidated. METHODS: Twenty-three participants with gestational diabetes underwent home sleep apnea testing. Obstructive sleep apnea was defined as an apnea hypopnea index > 5. Fasting morning blood samples were measured using multianalyte profiling (xMAP) multiplexed bead array immunoassay for Interleukin 6, tumor necrosis factor-alpha, and Interleukin 8. RESULTS: Age, body mass index, and gestational age at enrollment were 31 + 4.4 years, 35.7 + 7.4 kg/m2, and 28 ± 4 weeks, respectively. Participants were 52% Caucasian and 16% had obstructive sleep apnea. We observed positive correlations between apnea hypopnea index and Interleukin 6 (r = 0.62, p = 0.005), Interleukin 8 (r = 0.56, p = .56), and tumor necrosis factor-alpha (r = .58, p = .009). Women with obstructive sleep apnea had higher levels of Interleukin 6 (F = 5.01, p = .037) and Interleukin 8 (F = 6.33, p = .021) vs. women without obstructive sleep apnea. CONCLUSION: These preliminary results indicate that in women with gestational diabetes, apnea hypopnea index is associated with an elevated inflammatory profile.

Therapeutic Advantages of Dual Targeting of PPAR-δ and PPAR-γ in an Experimental Model of Sporadic Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is associated with progressive impairments in brain responsiveness to insulin and insulin-like growth factor (IGF). Although deficiencies in brain insulin and IGF could be ameliorated with trophic factors such as insulin, impairments in receptor expression, binding, and tyrosine kinase activation require alternative strategies. Peroxisome proliferator-activated receptor (PPAR) agonists target genes downstream of insulin/IGF stimulation. Furthermore, their anti-oxidant and anti-inflammatory effects address other pathologies contributing to neurodegeneration. OBJECTIVES: The goal of this research was to examine effects of dual delivery of L165, 041 (PPAR-δ) and F-L-Leu (PPAR-γ) agonists for remediating in the early stages of neurodegeneration. MODEL: Experiments were conducted using frontal lobe slice cultures from an intracerebral Streptozotocin (i.c. STZ) rat model of AD. RESULTS: PPAR-δ+ PPAR-γ agonist treatments increased indices of neuronal and myelin maturation, and mitochondrial proliferation and function, and decreased neuroinflammation, AßPP-Aß, neurotoxicity, ubiquitin, and nitrosative stress, but failed to restore choline acetyl transferase expression and adversely increased HNE(lipid peroxidation) and acetylcholinesterase, which would have further increased stress and reduced cholinergic function in the STZ brain cultures. CONCLUSION: PPAR-δ + PPAR-γ agonist treatments have substantial positive early therapeutic targeting effects on AD-associated molecular and biochemical brain pathologies. However, additional or alternative strategies may be needed to optimize disease remediation during the initial phases of treatment.

The rise and fall of insulin signaling in Alzheimer's disease.

The prevalence of both diabetes and Alzheimer's disease (AD) are reaching epidemic proportions worldwide. Alarmingly, diabetes is also a risk factor for Alzheimer's disease. The AD brain is characterised by the accumulation of peptides called Aß as plaques in the neuropil and hyperphosphorylated tau protein in the form of neurofibrillary tangles within neurons. How diabetes confers risk is unknown but a simple linear relationship has been proposed whereby the hyperinsulinemia associated with type 2 diabetes leads to decreased insulin signaling in the brain, with downregulation of the PI3K/AKT signalling pathway and its inhibition of the major tau kinase, glycogen synthase kinase 3ß. The earliest studies of post mortem AD brain tissue largely confirmed this cascade of events but subsequent studies have generally found either an upregulation of AKT activity, or that the relationship between insulin signaling and AD is independent of glycogen synthase kinase 3ß altogether. Given the lack of success of beta-amyloid-reducing therapies in clinical trials, there is intense interest in finding alternative or adjunctive therapeutic targets for AD. Insulin signaling is a neuroprotective pathway and represents an attractive therapeutic option. However, this incredibly complex signaling pathway is not fully understood in the human brain and particularly in the context of AD. Here, we review the ups and downs of the research efforts aimed at understanding how diabetes modifies AD risk.

Enzymatic Responses to Alcohol and Tobacco Nicotine-Derived Nitrosamine Ketone Exposures in Long Evans Rat Livers.

BACKGROUND: Chronic feeding plus binge administration of ethanol causes very high blood alcohol concentrations. However, its co-administration with tobacco Nicotine-Derived Nitrosamine Ketone (NNK) results in somewhat lower blood alcohol levels, suggesting that NNK and therefore smoking, alters alcohol metabolism in the liver. To explore this hypothesis, we examined effects of ethanol and/or NNK exposures on the expression and activity levels of enzymes that regulate their metabolism in liver. METHODS: This study utilized a 4-way model in which Long Evans rats were fed liquid diets containing 0% or 26% ethanol for 8 weeks, and respectively i.p injected with saline or 2 g/kg of ethanol 3 times/week during Weeks 7 and 8. The control and ethanol-exposed groups were each sub-divided and further i.p treated with 2 mg/kg of NNK or saline (3×/week) in Weeks 3-8. ADH, catalase and ALDH activities were measured using commercial kits. CYP450 mRNA levels (17 isoforms) were measured by qRT-PCR analysis. RESULTS: Ethanol significantly increased hepatic ADH but not catalase or ALDH activity. NNK had no effect on ADH, ALDH, or catalase, but when combined with ethanol, it increased ADH activity above the levels measured in all other groups. Ethanol increased CYP2C7, while NNK increased CYP2B1 and CYP4A1mRNA levels relative to control. In contrast, dual ethanol + NNK exposures inhibited CYP2B1 and CYP4A1 expression relative to NNK. Conclusion: Dual exposures to ethanol and NNK increase hepatic ethanol metabolism, and ethanol and/or NNK exposures alter the expression of CYP450 isoforms that are utilized in NNK and fatty acid metabolism.

Leptin levels are reduced by intravenous ghrelin administration and correlated with cue-induced alcohol craving.

Increasing evidence supports the role of appetite-regulating pathways, including ghrelin and leptin, in alcoholism. This study tested the hypothesis that intravenous exogenous ghrelin administration acutely decreases endogenous serum leptin levels, and that changes in leptin levels negatively correlate with alcohol craving. This was a double-blind, placebo-controlled human laboratory study. Non-treatment-seeking, alcohol-dependent, heavy drinkers (n=45) were randomized to receive intravenous ghrelin or placebo, followed by a cue-reactivity procedure, during which participants were exposed to neutral (juice) and alcohol trial cues. There was a main effect for intravenous ghrelin administration, compared with placebo, in reducing serum leptin levels (P<0.01). Post hoc analysis showed significant differences in serum leptin levels at the alcohol trial (P<0.05) that persisted at the end of the experiment (P<0.05). By contrast, there were no significant differences in serum leptin levels at the juice trial (P=not significant (NS)). The change of serum leptin level at the alcohol trial correlated with the increase in alcohol urge (P<0.05), whereas urge to drink juice was not correlated with the leptin change at the juice trial (P=NS). These findings provide preliminary evidence of ghrelin-leptin cross-talk in alcoholic individuals and suggest that their relationship may have a role in alcohol craving.

Dose effect of gestational ethanol exposure on placentation and fetal growth.

INTRODUCTION: Prenatal ethanol exposure compromises fetal growth by impairing placentation. Invasive trophoblastic cells, which mediate placentation, express the insulin-IGF regulated gene, aspartyl-asparaginyl ß-hydroxylase (ASPH), which has a critical role in cell motility and invasion. The aims of this study were to characterize effects of ethanol on trophoblastic cell motility, and assess ethanol dose-dependent impairments in placentation and fetal development. METHODS: Pregnant Long Evans dams were fed with isocaloric liquid diets containing 0%, 8%, 18% or 37% ethanol (caloric content) from gestation day (GD) 6 to GD18. Fetal development, placental morphology, density of invasive trophoblasts at the mesometrial triangle, as well as placental and mesometrial ASPH and Notch-1 protein expression were evaluated. Directional motility of control and ethanol-exposed HTR-8/SVneo cells was assessed by ATP Luminescence-Based assay. RESULTS: Severity of fetal growth impairment correlated with increasing doses of ethanol. Ethanol exposure produced dose-dependent alterations in branching morphogenesis at the labyrinthine zone, and inhibited physiological transformation of maternal arteries. ASPH and Notch-1 protein expression levels were reduced, corresponding with impairments in placentation. DISCUSSION: Prenatal ethanol exposure compromises fetal growth and placentation in a dose-responsive manner. Ethanol's adverse effects on placental development are mediated by: (1) altered branching morphogenesis in labyrinthine zone; (2) suppression of invasive trophoblastic precursor cells; and (3) inhibition of trophoblastic cell adhesion and motility, corresponding with reduced ASPH and Notch-1 protein expression.

Metabolic derangements mediate cognitive impairment and Alzheimer's disease: role of peripheral insulin-resistance diseases.

Herein, we review evidence that systemic insulin-resistance diseases linked to obesity, type 2 diabetes, and non-alcoholic steatohepatitis promote neurodegeneration. Insulin-resistance dysregulates lipid metabolism, which promotes ceramide accumulation with attendant inflammation and endoplasmic reticulum (ER) stress. Mechanistically, we propose that toxic ceramides generated in extra-CNS tissues, e.g. liver, get released into peripheral blood, and subsequently transit across the blood-brain barrier into the brain where they induce brain insulin-resistance, inflammation, and cell death (extrinsic pathway). These abnormalities establish or help propagate a cascade of neurodegeneration associated with increased ER stress and ceramide generation, which exacerbate brain insulin-resistance, cell death, myelin degeneration, and neuro-inflammation. The data suggest that a mal-signaling network mediated by toxic ceramides, ER stress, and insulin-resistance should be targeted to disrupt positive feedback loops that drive the AD neurodegeneration cascade.

Precision-cut slice culture method for rat placenta.

Primary trophoblasts, placental explants, and cell line cultures are commonly used to investigate placental development, physiology, and pathology, particularly in relation to pregnancy outcomes. Organotypic slice cultures are increasingly used in other systems because they maintain the normal three-dimensional tissue architecture and have all cell types represented. Herein, we demonstrate the utility of the precision-cut placental slice culture model for studying trophoblastic diseases.

Impaired placentation in fetal alcohol syndrome.

Intrauterine growth restriction (IUGR) is one of the key features of fetal alcohol syndrome (FAS), and IUGR can be mediated by impaired placentation. Insulin-like growth factors (IGF) regulate placentation due to stimulatory effects on extravillous trophoblasts, which are highly motile and invasive. Previous studies demonstrated that extravillous trophoblasts express high levels of aspartyl-(asparaginyl) beta-hydroxylase (AAH), a gene that is regulated by IGF and has a critical role in cell motility and invasion. The present study examines the hypothesis that ethanol impaired placentation is associated with inhibition of AAH expression in trophoblasts. Pregnant Long Evans rats were fed isocaloric liquid diets containing 0% or 37% ethanol by caloric content. Placentas harvested on gestation day 16 were used for histopathological, mRNA, and protein studies to examine AAH expression in relation to the integrity of placentation and ethanol exposure. Chronic ethanol feeding prevented or impaired the physiological conversion of uterine vessels required for expansion of maternal circulation into placenta, a crucial process for adequate placentation. Real-time quantitative RT-PCR analysis demonstrated significant reductions in IRS-1, IRS-2, and significant increases in IGF-II and IGF-II receptor mRNA levels in ethanol-exposed placentas. These abnormalities were associated with significantly reduced levels of AAH expression in trophoblastic cells, particularly within the mesometrial triangle (deep placental bed) as demonstrated by real time quantitative RT-PCR, Western blot analysis, ELISA, and immunohistochemical staining. Ethanol-impaired placentation is associated with inhibition of AAH expression in trophoblasts. This effect of chronic gestational exposure to ethanol may contribute to IUGR in FAS.

Chronic gestational exposure to ethanol causes insulin and IGF resistance and impairs acetylcholine homeostasis in the brain.

In fetal alcohol syndrome (FAS), cerebellar hypoplasia is associated with impaired insulin-stimulated survival signaling. This study characterizes ethanol dose-effects on cerebellar development, expression of genes required for insulin and insulin-like growth factor (IGF) signaling, and the upstream mechanisms and downstream consequences of impaired signaling in relation to acetylcholine (ACh) homeostasis. Pregnant Long Evans rats were fed isocaloric liquid diets containing 0%, 2%, 4.5%, 6.5%, or 9.25% ethanol from gestation day 6. Ethanol caused dose-dependent increases in severity of cerebellar hypoplasia, neuronal loss, proliferation of astrocytes and microglia, and DNA damage. Ethanol also reduced insulin, IGF-I, and IGF-II receptor binding, insulin and IGF-I receptor tyrosine kinase activities, ATP, membrane cholesterol, and choline acetyltransferase (ChAT) expression. In vitro studies linked membrane cholesterol depletion to impaired insulin receptor binding and insulin-stimulated ChAT. In conclusion, cerebellar hypoplasia in FAS is mediated by insulin/IGF resistance with attendant impairments in energy production and ACh homeostasis.

Ethanol inhibits insulin expression and actions in the developing brain.

Ethanol-induced cerebellar hypoplasia is associated with inhibition of insulin-stimulated survival signaling. The present work explores the mechanisms of impaired insulin signaling in a rat model of fetal alcohol syndrome. Real-time quantitative RT-PCR demonstrated reduced expression of the insulin gene in cerebella of ethanol-exposed pups. Although receptor expression was unaffected, insulin and insulin-like growth factor (IGF-I) receptor tyrosine kinase (RTK) activities were reduced by ethanol exposure, and these abnormalities were associated with increased PTP1b activity. In addition, glucose transporter molecule expression and steady-state levels of ATP were reduced in ethanol-exposed cerebellar tissue. Cultured cerebellar granule neurons from ethanol-exposed pups had reduced expression of genes encoding insulin, IGF-II, and the IGF-I and IGF-II receptors, and impaired insulin- and IGF-I-stimulated glucose uptake and ATP production. The results demonstrate that ethanol inhibits insulin-mediated actions in the developing brain by reducing local insulin production and insulin RTK activation, leading to inhibition of glucose transport and ATP production.

Neuronal thread protein regulation and interaction with microtubule-associated proteins in SH-Sy5y neuronal cells.

In Alzheimer's disease (AD), neuronal thread protein (NTP) accumulates in cortical neurons and colocalizes with phospho- tau-immunoreactive cytoskeletal lesions that correlate with dementia. To generate additional information about the potential role of NTP in AD, we characterized its expression and regulation in human SH-Sy5y neuronal cells. Quantitative real-time reverse transcription-polymerase chain reactin and Western blot analysis demonstrated prominent insulin, moderate insulin-like growth factor, type 1 (IGF-1) and minimal nerve growth factor stimulation of NTP expression. In addition, NTP protein was more stable and it progressively accumulated in cells that were stimulated with insulin for 24 or 48 h. Metabolic labeling and phospho-amino acid analysis demonstrated phosphorylation of NTP on Serine residues, 30-60 min after insulin or IGF-1 stimulation, when glycogen synthase kinase 3beta (GSK-3beta) activity would no longer have been suppressed. Kinase inhibitor and in vitro phosphorylation studies demonstrated a role for GSK-3beta in the positive regulation of NTP expression and phosphorylation. Coimmunoprecipitation studies demonstrated physical interactions between NTP and tau or microtubule-associated protein 1b (MAP-1b), and ubiquitin immunoreactivity in NTP immunoprecipitates. In summary, these studies showed that (i) NTP expression is regulated at the level of transcription by insulin and IGF-1 stimulation; (ii) NTP is phosphorylated by GSK-3beta; (iii) NTP can physically interact with tau and MAP-1b and (iv) NTP-MAP complexes are ubiquitinated. The results suggest a functional role for NTP in relation to the turnover or processing of neuronal cytoskeletal proteins, attributes that may be modulated by insulin/IGF-1-mediated signaling.

Chronic gestational exposure to ethanol impairs insulin-stimulated survival and mitochondrial function in cerebellar neurons.

Chronic gestational exposure to ethanol has profound adverse effects on brain development. In this regard, studies using in vitro models of ethanol exposure demonstrated impaired insulin signaling mechanisms associated with increased apoptosis and reduced mitochondrial function in neuronal cells. To determine the relevance of these findings to fetal alcohol syndrome, we examined mechanisms of insulin-stimulated neuronal survival and mitochondrial function using a rat model of chronic gestational exposure to ethanol. In ethanol-exposed pups, the cerebellar hemispheres were hypoplastic and exhibited increased apoptosis. Isolated cerebellar neurons were cultured to selectively evaluate insulin responsiveness. Gestational exposure to ethanol inhibited insulin-stimulated neuronal viability, mitochondrial function, Calcein AM retention (membrane integrity), and GAPDH expression, and increased dihydrorosamine fluorescence (oxidative stress) and pro-apoptosis gene expression (p53, Fas-receptor, and Fas-ligand). In addition, neuronal cultures generated from ethanol-exposed pups had reduced levels of insulin-stimulated Akt, GSK-3beta, and BAD phosphorylation, and increased levels of non-phosphorylated (activated) GSK-3beta and BAD protein expression. The aggregate results suggest that insulin-stimulated central nervous system neuronal survival mechanisms are significantly impaired by chronic gestational exposure to ethanol, and that the abnormalities in insulin signaling mechanisms persist in the early postnatal period, which is critical for brain development.

Neurodegeneration changes in primary central nervous system neurons transfected with the Alzheimer-associated neuronal thread protein gene.

The AD7c-NTP gene is over-expressed in brains with Alzheimer's disease (AD), and increased levels of the corresponding protein are detectable in cortical neurons, brain tissue extracts, cerebrospinal fluid, and urine beginning early in the course of AD neurodegeneration. In the present study, we utilized a novel method to transfect post-mitotic primary neuronal cell cultures, and demonstrated that over-expression of the AD7c-NTP gene causes cell death and neuritic sprouting, two prominent abnormalities associated with AD. These results provide further evidence that aberrantly increased AD7c-NTP expression may have a role in AD-type neurodegeneration. In addition, we demonstrate that primary post-mitotic neurons can be efficiently transfected with conventional recombinant plasmid DNA to evaluate the effects of gene over-expression in relevant in vitro models.

Mitochondrial dna damage and impaired mitochondrial function contribute to apoptosis of insulin-stimulated ethanol-exposed neuronal cells.

BACKGROUND: Ethanol inhibition of insulin signaling may contribute to impaired central nervous system development in fetal alcohol syndrome. An important consequence of ethanol inhibition of insulin signaling is increased apoptosis due to reduced levels of insulin-stimulated phosphoinositol-3-kinase activity. METHODS: We used viability assays, end-labeling, Western blot analysis, and MitoTracker (Molecular Probes, Eugene, OR) fluorescence labeling to determine whether ethanol-induced central nervous system neuronal cell death was mediated in part by increased mitochondrial (Mt) DNA damage and impaired Mt function. RESULTS: In ethanol-exposed, insulin-stimulated PNET2 central nervous system-derived human neuronal cells, reduced viability was associated with increased Mt DNA damage, reduced Mt mass (manifested by reduced Mt protein expression and MitoTracker Green fluorescent labeling), and impaired Mt function (manifested by reduced levels of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide activity, cytochrome oxidase-Complex IV, Subunit II expression, and MitoTracker Red fluorescence). The adverse effects of ethanol on Mt function were reduced by pretreating the cells with broad-spectrum caspase inhibitors and nearly abolished by nerve growth factor stimulation, with or without concomitant treatment with global caspase inhibitors. CONCLUSIONS: These results suggest that ethanol-induced death of insulin-stimulated immature neuronal cells is mediated in part by impaired Mt function associated with Mt DNA damage and reduced Mt mass, and therefore it is likely to contribute to neuronal loss associated with fetal alcohol syndrome. The findings also suggest that the adverse effects of ethanol on insulin-stimulated survival and metabolic function could be overcome by stimulating with growth factors that support Mt function through insulin-independent pathways.

Alzheimer-associated neuronal thread protein-induced apoptosis and impaired mitochondrial function in human central nervous system-derived neuronal cells.

In Alzheimer Disease (AD), dementia is due to cell loss and impaired synaptic function. The cell loss is mediated by increased apoptosis, predisposition to apoptosis, and impaired mitochondrial function. Previous studies demonstrated that the AD7c-NTP neuronal thread protein gene is over-expressed in AD beginning early in the course of disease, and that in AD, AD7c-NTP protein accumulation in neurons co-localizes with phospho-tau-immunoreactivity. To determine the potential contribution of AD7c-NTP over-expression to cell loss in AD, we utilized an inducible mammalian expression system to regulate AD7c-NTP gene expression in human CNS-derived neuronal cells by stimulation with isopropyl-1-beta-D-thiogalactopyranoside (IPTG). IPTG induction of AD7c-NTP gene expression resulted in increased cell death mediated by apoptosis, impaired mitochondrial function, and increased cellular levels of the p53 and CD95 pro-apoptosis gene products as occur in AD. In addition, over-expression of AD7c-NTP was associated with increased levels of phospho-tau, but not amyloid-beta immunoreactivity. These results suggest that AD7c-NTP over-expression may have a direct role in mediating some of the important cell death cascades associated with AD neurodegeneration, and further establish a link between AD7c-NTP overexpression and the accumulation of phospho-tau in preapoptotic CNS neuronal cells.

Ethanol impairs insulin-stimulated mitochondrial function in cerebellar granule neurons.

Ethanol impairs insulin-stimulated survival and mitochondrial function in immature proliferating neuronal cells due to marked inhibition of downstream signaling through P13 kinase. The present study demonstrates that, in contrast to immature neuronal cells, the major adverse effect of chronic ethanol exposure (50 mM) in post-mitotic rat cerebellar granule neurons is to inhibit insulin-stimulated mitochondrial function (MTT activity, MitoTracker Red fluorescence, and cytochrome oxidase immunoreactivity). Ethanol-impaired mitochondrial function was associated with increased expression of the p53 and CD95 pro-apoptosis genes, reduced Calcein AM retention (a measure of membrane integrity), increased SYTOX Green and propidium iodide uptake (indices of membrane permeability), and increased oxidant production (dihydrorosamine fluorescence and H2O2 generation). The findings of reduced membrane integrity and mitochondrial function in short-term (24 h) ethanol-exposed neurons indicate that these adverse effects of ethanol can develop rapidly and do not require chronic neurotoxic injury. A role for caspase activation as a mediator of impaired mitochondrial function was demonstrated by the partial rescue observed in cells that were pre-treated with broad-spectrum caspase inhibitors. Finally, we obtained evidence that the inhibitory effects of ethanol on mitochondrial function and membrane integrity were greater in insulin-stimulated compared with nerve growth factor-stimulated cultures. These observations suggest that activation of insulin-independent signaling pathways, or the use of insulin sensitizer agents that enhance insulin signaling may help preserve viability and function in neurons injured by gestational exposure to ethanol.

Oxidative stress and hypoxia-like injury cause Alzheimer-type molecular abnormalities in central nervous system neurons.

Neuronal loss and neuritic/cytoskeletal lesions (synaptic disconnection and proliferation of dystrophic neurites) represent major dementia-associated abnormalities in Alzheimer's disease (AD). This study examined the role of oxidative stress as a factor contributing to both the cell death and neuritic degeneration cascades in AD. Primary neuron cultures were treated with H2O2 (9-90 microM) or desferrioxamine (2-25 microM) for 24 h and then analyzed for viability, mitochondrial mass, mitochondrial function, and pro-apoptosis and sprouting gene expression. H2O2 treatment causes free-radical injury and desferrioxamine causes hypoxia-type injury without free radical generation. The H2O2-treated cells exhibited sustained viability but neurite retraction, impaired mitochondrial function, increased levels of the pro-apoptosis gene product CD95/Fas, reduced expression of N2J1-immunoreactive neuronal thread protein and synaptophysin, and reduced distribution of mitochondria in neuritic processes. Desferrioxamine treatment resulted in dose-dependent neuronal loss associated with impaired mitochondrial function, proliferation of neurites, and reduced expression of GAP-43, which has a role in path-finding during neurite outgrowth. The results suggest that oxidative stress can cause neurodegeneration associated with enhanced susceptibility to apoptosis due to activation of pro-apoptosis genes, neurite retraction (synaptic disconnection), and impaired transport of mitochondria to cell processes where they are likely required for synaptic function. In contrast, hypoxia-type injury causes neuronal loss with proliferation of neurites (sprouting), impaired mitochondrial function, and reduced expression of molecules required to form and maintain synaptic connections. Since similar abnormalities occur in AD, both oxidative stress and hypoxic injury can contribute to AD neurodegeneration.